Durable anti-microbial agents

ABSTRACT

The present invention comprises a metal complex capable of being dispersed in or alternatively, formed in a water-absorbing article. The metal complex consists of one or more metal ions, at least one chelating polymer chelated to the transition metal ion and at least one potentiator chelated to the transition metal ion. The chelating polymer may be a polyglucosamine, a polycarboxylic acid, a polyamine and methylene-acrylic acid polymer.

This is a division of application No. 08/301,043 filed Sep. 6, 1994, nowU.S. Pat. No. 5,541,233, which is a continuation of 07/983,685, filedDec. 1, 1992, now abandoned.

FIELD OF INVENTION

The present invention relates to anti-microbial agents. In particular,the present invention relates to a sponge containing anti-microbialagents.

BACKGROUND OF THE INVENTION

Sponges are light, fibrous connective structures which have absorbentqualities. They can be made from a variety of different materialsincluding polymers such as urethanes and cellulose in a number ofdifferent ways.

A preferred sponge is the cellulose sponge because of its excellentwater sorption qualities. These sponges are made by dispersing sodiumsulfate crystals in a viscose cellulose. Once mixed with the viscosecellulose, the sodium sulfate crystals are melted out of the sponge byheating the viscose cellulose while the viscose cellulose is regeneratedor coagulated to an insoluble state. Once regenerated, the viscosecellulose sponge is rinsed.

Although exhibiting excellent water sorption qualities superior to othersponges, cellulose sponges have several drawbacks due to thischaracteristic. One drawback is that sponges can absorb moisturecontaining unwanted microorganisms such as bacteria and fungi. Thesemicroorganisms thrive and rapidly multiply in the moist environmentfound in the sponge and can degrade the sponge by causing loss in spongestrength and integrity. In addition, the microorganisms can emit an odorwhich is unpleasant to a sponge user.

Moreover, many of these microorganisms are pathogenic thereby raisinghealth and safety concerns. Pathogenic microorganisms such as gramnegative bacteria, gram positive bacteria, yeast and fungi have all beenfound in sponges. Such concerns are especially relevant in the foodservice and medical industries where sanitization and the prevention ofthe spread infectious disease is of utmost importance. For example, inthe food service industry, salmonella choleraesuis can be transferred toa sponge from a surface contacted by the sponge. The salmonellacholeraesuis can then multiply in the sponge and be transferred toanother surface thereby increasing the chance of infection.

Several attempts have been made to control or prevent the growth ofthese unwanted microorganisms in cellulosic sponges by treating thesponges with anti-microbial agents such as biocides. For example, U.S.Pat. No. 3,018,192 ("Hennemann et al.") discloses the use of a reactionproduct of a quaternary ammonium compound with either an alkali metalsalt or carboxymethyl cellulose as the biocide.

U.S. Pat. No. 3,594,221 ("Baldwin") describes another process oftreating fibrous materials with a germicide such as a quaternaryammonium compound. In this process, the fibrous materials are firstimpregnated with an acid or alkali metal montmorillonite clay. Onceimpregnated with the clay, the materials are infused with the germicide.

Another attempt to prevent microorganism growth in sponges and othercellulose-based products is described in EPO No. 358,572 ("Coilin").Collin discloses a post-regenerative treatment imparting biocidalproperties to porous cellulose-based products. The cellulose-basedproducts are impregnated with a solution containing an aqueous biocidesuch as a quaternary ammonium compound or an oligomericpolyalkylene-2-guanide salt. The solution also contains a binder such asan acrylic latex, butadiene-styrene latex or vinylic latex. Afterimpregnation, a second solution is contacted with the cellulose-basedproduct. This second solution precipitates the biocidal agent onto theporous surfaces of the cellulose-based product and in addition,coagulates the binder.

A different biocidal sponge treatment described in U.S. Pat. No.3,586,520. ("Dillon"). Dillon teaches that metal dialkyldithiocarbamates can be utilized as biocides in pigmented sponges.

Although many cellulose sponges treated with anti-microbial agentsexhibit biocidal activity, many such cellulose sponges have one or moreof the following drawbacks. Some treatments impact upon the viscosexanthation, regeneration and/or subsequent rinsing processes commonlyutilized in sponge manufacture. Conversely, the manufacturing processmay affect the treatment rendering it ineffectual. Some anti-microbialtreatments adversely affect the feel, color, flexibility, texture orwater sorption properties of the sponges. In other sponges, the biocidalactivity is short-lived due to numerous washings that the spongeexperiences during its product life. Yet, other sponges pose a toxicthreat to both the environment and to humans.

Thus, there currently exists a need for an anti-microbial agent which iscapable of being dispersed or formed in a water absorbing article whichcan maintain long-lasting anti-microbial activity. There also exists aneed for an anti-microbial agent which eliminates many adverse toxiceffects to humans and the environment while maintaining long-lastinganti-microbial activity. In addition, there is a need for aanti-microbial treatment of a sponge wherein the treatment does notadversely affect the feel, color, odor, flexibility or texture of thearticle nor does it impact upon the xanthation, regeneration, viscose orrinsing processes commonly utilized in sponge manufacture.

SUMMARY OF THE INVENTION

The present invention comprises a metal complex which exhibitsanti-microbial activity and which is capable of being dispersed orformed within a water-absorbing porous article. The metal complexcomprises one or more chelating polymers, at least one transition metalion and at least one anti-microbial potentiator.

A useful embodiment of the present invention comprises a water-absorbingporous article such as a sponge possessing durable anti-microbialactivity and exhibiting minimal toxic effects to humans and theenvironment. The porous article of the present invention is notadversely affected by the anti-microbial complex nor does it adverselyaffect the anti-microbial activity of the complex.

The present invention also comprises a method of making thewater-absorbing articles of the present invention.

DETAILED DESCRIPTION

The anti-microbial agent of the present invention comprises one or morechelating polymers, at least one transition metal ion and anti-microbialpotentiator. This agent is capable of being dispersed or formed in apolymeric or water-absorbing article such as a sponge.

Transition metal ions are the ions of the metals classified in groups IBto VIIIB of the periodic table of elements. The metal elements aregenerally characterized as in having incomplete inner rings of electronsor being otherwise capable of existing in more than one valence state.The transition metals can exist in aqueous solution as cations and formionic/covalent bonds with other species generally referred to aschelating agents or ligands. Examples of suitable metal ions are thosethat have coordination numbers between 2 and 8. Suitable transitionmetal ions include zinc, zirconium, copper and aluminum.

The transition metals of the present invention form a coordinate bondwith ligands referred to as chelating polymers. It is theorized that thechelating polymers donate electrons to the ionic transition metal ion.Suitable chelating polymers include any polymer containing functionalgroups in close proximity capable of donating electrons. Examples ofchelating polymers which can be utilized in the present inventioninclude, but are not limited to, polyglucosamines, ethylene acrylic acidcopolymers, polycarboxylic acids or polyamines. Preferred polymersinclude those which can have hydroxyl and/or amine groups. An especiallypreferred chelating polymer is the glucosamine chitosan. Chitosan is thedeacetylated derivative of the polysaccharide chitin[β-(1,4)-poly-N-acetyl-D glucosamine], an abundant natural by-product ofthe shrimp and crab industries.

In addition to chelating with the chelating polymers, the transitionmetal ions are also chelated to potentiators. For the purposes of thisapplication, a potentiator is defined as an anti-microbial agent capableof chelating to a transition metal ion. It should be noted that theselection of the potentiators is dependent upon the coordinationchemistry of the metal ion. If the metal ion can be completely displacedby a potentiator, the durability of the complex within the chelatingpolymer can be compromised and thus, use of such potentiator is notdesirable for durability purposes. Suitable potentiators include, butare not limited to, alkyl dithiocarbamates, thiazoles, imidazoles andpyrithiones.

Suitable alkyl dithiocarbamates are those wherein each alkyl group ofthe carbamate has up to about eight carbons. The alkyl groups can bestraight or branched. Representative dithiocarbamates include dimethyldithiocarbamate, diethyl dithiocarbamate, dipropyl dithiocarbamate,dibutyl dithiocarbamate, methyl ethyl dithiocarbamate, methyl propyldithiocarbamate, methyl butyl dithiocarbamate, dihexyl dithiocarbamate,dioctyl dithiocarbamate and the like.

Imidazoles are a five-membered heterocylic compound which have thefollowing structure: ##STR1## An example of a suitable imidazole is2-(4-thiazolyl) benzimidazole.

Thiazoles are five-membered rings containing nitrogen and sulfur in theone and three positions respectively. An example of a suitable thiazoleis 2-mercaptobenzothiazole.

An especially preferred potentiator is pyrithione which has thefollowing structure: ##STR2## Pyrithiones are sold as zinc or sodiumsalts which can be obtained from the Olin Corporation under thetradename OMADINE.

If desired, natural or synthetic reinforcing fibers can be added to thearticle of the present invention. Suitable natural reinforcing fibersinclude cotton, flax, hemp, ramie, rayon, burlap, shoddy cotton, cottonlinters and pulp fibers. Representative examples of synthetic fibersinclude polyester, nylon and acrylic fibers.

Additives can be included in the mixture to enhance specific objectives.For example, pigments, pigment fixing agents and processing aids can beadded.

The sponge of the present invention can be a cellulose sponge which isprepared from viscose cellulose. The viscose cellulose can be made fromany conventional viscose technique. Briefly, the viscose cellulose iscommonly prepared through the mercerization and shredding of wood pulp,followed by xanthation with carbon disulfide, dilution with water andfinally, mixing the mixture. After the viscose cellulose is made,crystals of sodium sulfate decahydrate, commonly referred to asGlauber's Salt, are added to the viscose cellulose. The chelatingpolymer and optional reinforcing fibers and/or addititives are thenadded. The transition metal ion, alone or in combination with thepotentiator may be added to the mixture at this time although it is notnecessary. After mixing the ingredients, the mixture is heated in acontrolled fashion to approximately 100° C. by conductive or R.F.heating. Such heat treatment coagulates and regenerates the cellulosewhile melting the sodium sulfate away. The sodium sulfate is thendrained and rinsed from the resultant regenerated sponge leaving aporous structure. Finally, the potentiator or transition metal ion incombination with the potentiator, if not added previously, is introducedinto the article.

The sponge of the present invention can comprise from about 10 to about90 weight percent viscose cellulose, more preferably from about 20 to 80weight percent viscose cellulose. If added, the reinforcing fibers cancomprise up to 90 weight percent of the sponge, preferably, 20 to 80weight percent of the sponge. The amount of chelating polymer dependsupon the amount of viscose cellulose added. Generally, the chelatingpolymer will comprise from about 0.01 to about 50 weight percent of theviscose cellulose. More preferably, the chelating polymer will comprisefrom about 0.1 to about 20 weight percent of the viscose cellulose. Thetransition metal ion may comprise from about 0.1 to about 50 weightpercent of the chelating polymer and more preferably, should comprisefrom about 1 to about 30 weight percent of the chelating polymer. Theultimate amount of potentiator depends upon the amount of anti-microbialactivity that the user wants to impart to the sponge. However, themaximum chelatable amount of the potentiator is fixed by thecoordination number and/or weight percent of the transition metal ionand will therefore, preferentially lie in the range of one to four molesof potentiator per mole of transition metal ion present in the sponge.

The following examples are set forth to illustrate this invention andare not intended to limit the scope of this invention thereof.

EXAMPLES SUBSTANTIVITY OF METAL-CHITOSAN COMPLEX THROUGHOUT THE VISCOSESPONGE-MAKING PROCESS

PRECURSORs 1a-g

Chitosan was purchased from the Vanson Chemical Company, Redmond, Wash.,in two different grades as shown in the table below:

                  TABLE I                                                         ______________________________________                                        Chitosan Grade                                                                              VNS-461   VNS-457                                               ______________________________________                                        MW (approx)     50,000      100,000                                           Insolubles      0.23        0.68                                              Deacetylation   92.3%       76.4%                                             Ash             0.12        0.2                                               ______________________________________                                    

Zinc sulfate solution was prepared by dissolving 5.5 g zinc sulfateheptahydrate in 89.5 ml deionized (DI) water. Copper sulfate solutionwas prepared by dissolving 4.9 g copper sulfate pentahydrate in 90.1 mldeionized water. Approximately 5 g chitosan was added to each metal saltsolution, which was stirred approximately 30 minutes to disperse thechitosan, and the resulting chitosan-metal complex was filtered bysuction. Each chitosan-metal filter cake was:

a) rinsed on the filter with 100 ml tap water;

b) slurried in 150 ml 1.5% NaOH, boiled 45 minutes, filtered and rinsedwith 100 ml DI water; and

c) rinsed on the filter with 0.3% sodium hypochlorite solution(household bleach diluted 1:15 with water) followed by a rinse with 500ml DI water.

After each of the steps a, b, c above, samples of the filter cakes weretaken for subsequent analysis. Table II below identifies each sample ofchitosan-metal complex according to chitosan grade, metal ion, andprocess step:

                  TABLE II                                                        ______________________________________                                                   Metal/Chitosan:                                                    Process Step CU/461   Cu/457   Zn/461 Zn/457                                  ______________________________________                                        a)  Filter cake  1a       1b     1c     --                                        water rinse:                                                              b)  Filter cake  1d       --     1e     --                                        boiling in                                                                    NaOH + water                                                                  rinse:                                                                    c)  Filter cake (b)                                                                            1f       --     1g     --                                        bleach rinse +                                                                water rinse:                                                              ______________________________________                                    

Filter cake samples were then dried in an oven at 70°-76° C. (for 2hours). The metal and sulfur content of the samples was determined byInductively Coupled. Plasma (ICP) analysis after digestion of thesamples with acetic acid. The ICP analysis was conducted with a Model3580 ICP Atomic Emission Spectrometer sold by Fisons Instruments ofValencia, Calif. Confirmatory analysis for metal content was performedby digestion of the samples with nitric and sulfuric acids. The metalanalysis test results of the samples are reported in Table III.

                  TABLE III                                                       ______________________________________                                                         Analysis                                                     Precursor #        Cu       Zn                                                ______________________________________                                        1a                 11.63%   --                                                1b                 10.08    --                                                1c                 --       11%                                               1d                 12.72    --                                                1e                 --        9.78                                             1f                 12.69    --                                                1g                 --       10.41                                             VNS-461 (untreated)                                                                              <8 ppm   9 ppm                                             ______________________________________                                    

The test results indicate that the chitosan-metal complex is stable toviscose treatment. This is indicated by comparing the theoretical yieldto the actual yield. Prediction of the theoretical yield is described inthe following paragraph:

The chitosan glucosamine repeat unit is identical to the celluloserepeat unit except for an amine group at the C2 position. Thus, with aglucosamine repeat unit molecular weight of 157, the theoretical percentof chelated substance may be estimated depending upon the assumed moleratio of metal ion to glucosamine unit. For example, for a mole ratio"r" of a metal M having the molecular weight "W," the theoreticalpercent metal in chitosan may be calculated from the equation: %=100%r.W./(157+r.W.'), assuming all repeat units are available for chelationand W is the molecular weight of the chelated compound (e.g., Zn orZnSO₄). However, a characteristic typical of chitosan is its degree ofdeacetylation, as a result of the process from which it is made.Chitosan may have low (approximately 75%) to high (approximately 90%)degree of deacetylation. The degree of deacetylation "D" is essentiallythe percentage of repeat units present with --NH₂ functionality.Conversely, 100-D is the percentage of repeat units with --NHCOCH₃functionality. If it is assumed that only repeat units with --NH₂functionality may participate in chelation, then the equation predictingpercent theoretical metal becomes:

    %=100 ·r·D·W/[D·157+(1-D)187.2+r·D.multidot.W')

Thus, for a 1:1 mole ratio of metal to glucosamine with grade VNS-462chitosan having a D=92.3%, the theoretical percent metal for copper orzinc ranges from approximately 27%, assuming no accompanying sulfate orother ion is present, to 19%, assuming sulfate ion is present. The testresults reported in Table III indicate metal contents in the 10 to 13percent range thereby suggesting approximately a 50% efficiency relativeto the maximum chelation possible.

Moreover, the metal analysis results indicate that the chitosan-metalcomplex is stable to the viscose sponge process insofar as both copperand zinc metal content of the samples remained relatively constantthroughout the boiling caustic and bleach rinsing process.

RELATIVE DURABILITY OF CHITOSAN-PYRITHIONE AND CHITOSAN-METAL-PYRITHIONECOMPLEXES Comparative Example 2a, Precursors 2b-c and Example 2d

Chelation of chitosan is conveniently carried out by slurrying chitosanin solutions with the desired chelation agents. The following describesthe preparation of Comparative Example 2a, Precursors 2b-c and Example2d:

a. Preparation of Comparative Example 2a (chitosan-pyrithione complex):a chitosan slurry was prepared by stirring 1 g VNS-461 into 50 g DIwater. A solution of pyrithione acid (PA) was prepared in a separateflask by mixing 2.61 g 40% sodium pyrithione (NAP) solution obtained assodium Omadine ("Omadine" is a trademark of Olin Corp, Cheshire, Conn.),with 22.4 g DI water and stirring followed by 25.28 g 1% hydrochloricacid. By this method, 99% of the NaP is acidified. The PA solution wasadded to the chitosan slurry, the mixture stirred 30 minutes, and thechitosan-pyrithione complex was filtered. This process was repeated fourmore times to yield five filter cakes of chitosan-pyrithione complex.

b. Preparation of Precursors 2b-c (metal-chitosan complex): one gramVNS-461 was stirred into a solution comprising 1.59 g CuSO₄ ·5H₂ Odissolved in 98.4 g DI water. After stirring the slurry for 30 minutes,the chitosan-copper complex was filtered off. Similarly, a chitosan-zinccomplex was prepared from 1 g VNS-461 slurried into a solutioncomprising 1.83 g ZnSO₄ ·7H₂ O and 98.2 g DI water. In all, threechitosan-copper filter cakes (Precursor 2b) and six chitosan-zinc filtercakes (Precursor 2c) were prepared in this fashion.

c. Preparation of Example 2d (chitosan-zinc pyrithione complex): threeof the above prepared chitosan-zinc filter cakes were, in turn, washed(on the filter) with 400 ml DI water. Each filter cake was then slurriedinto a solution comprising 4.75 g 40% NaP in 96.2 g DI water. Thiscorresponds to a 2:1 mole ratio allowance of pyrithione: zinc assuming100% coordination of the chitosan/glucosamine groups by zinc ion.

After 30 minutes stirring, the chitosan-zinc pyrithione complex wasfiltered.

The durability of the these chitosan complexes was determined bysubjecting these complexes to typical end-use conditions such as wouldbe expected of a sponge or wiping article in household use. Each filtercake was rinsed with water, bleach or detergent as indicated in TableIV. The cakes were thereafter tested for metal content by ICP analysisas described in Precursors 1a-g. The test results are reported in TableIV.

                  TABLE IV                                                        ______________________________________                                                       Metal Analysis                                                 Filter Cake                                                                              Treatment Cu        Zn     S                                       ______________________________________                                        Pyrithione acid                                                                          a         <13 ppm   <4 ppm 2.70%                                   & chitosan                                                                    (Comparative                                                                  Example 2a)                                                                              b         <3 ppm    <2 ppm 2.40%                                              c         <9 ppm    ND     2.10%                                              d         <15 ppm   <34 ppm                                                                              0.88%                                              e         <12 ppm   <25 ppm                                                                              0.33%                                   Copper &   c         13.80%    ND*    0.52%                                   chitosan                                                                      (Precursor 2b)                                                                           d         14.50%    ND     0.74%                                              e         14.70%    ND     0.48%                                   Zinc & chitosan                                                                          c         <30 ppm   11.04% 0.85%                                   (Precursor 2c)                                                                           d         <52 ppm   12.08% 0.81%                                              e         <58 ppm   12.01% 0.83%                                   Zinc pyrithione                                                                          c         --        6.46%  6.15%                                   & chitosan                                                                    (Example 2d)                                                                             d         --        6.72%  4.06%                                              e         --        6.61%  6.34%                                   ______________________________________                                         *indicates below detection levels.                                       

"Treatments" are on-the-filter rinsing as described below:

a) No rinse

b) Rinsed with 200 ml DI water

c) Rinsed with 400 ml DI water

d) Rinsed sequentially with 400 ml DI water, 200 ml 0.5%household/chlorine bleach, 200 ml DI water.

e) Rinsed sequentially with 400 ml DI water, 200 ml Spic & Span solution(3.4 g Spic & Span dissolved in 196.6 ml DI water). Spic & Span is acommercial product of Procter and Gamble Cincinnati, Ohio).

The test results revealed that the chitosan-pyrithione acid complex isminimally durable to washing with water and much less durable to bleachor detergent rinsing based on the observed decrease in sulfur contentfrom greater than 2% to less than 1% after the latter rinsings. Thecopper and zinc levels remain relatively constant in the 11-15% rangeindicating that they are clearly durable to the rinse treatments. Due tothe uptake of the pyrithione ligand, the zinc pyrithione treatment showsgood durability to the rinsing, although a drop in sulfur content isnoted in bleaching, possibly as a result of pyrithione displacement. Thezinc level was also lowered relative to the zinc chelation cakes to 60to 70 percent of the original level which is in agreement withtheoretical predictions. For example, comparing water rinsed zinc caketo zinc-pyrithione cake, the drop from 11.5% to 6.5% zinc representsroughly a 40% reduction, in agreement with theory. This observation,taken together with the measured zinc-to-sulfur weight ratios close tounity suggests the pyrithione may be present as the doubly coordinatedzinc pyrithione form. Unexpectedly, the data indicate that fullycoordinated zinc pyrithione while also chelated to chitosan is stable torinsing. For example, it is known that zinc-pyrithione occurs naturallyas 2 moles of pyrithione chelated to one mole zinc. Zinc in this casehas a coordination number of four and pyrithione is bidentate. Theseresults indicate that if zinc interacts with two repeat units ofchitosan, as earlier data suggest, then pyrithione is likely interactingwith zinc in a monodentate fashion. The balance of zinc interaction withchitosan and pyrithione is apparently such that the entire complexexhibits stability to common rinsing. This would not be expected ifeither the interaction of zinc with pyrithione were weakened due to theapparent monodentate behavior, or if the interaction of zinc withpyrithione were strong enough to displace zinc from chitosan. In eithercase, it would be expected that either pyrithione or zinc pyrithionewould have otherwise been readily rinsed out of the sponge, as observed,for example, in the case of the chitosan-pyrithione complex.

Note also, for a 1:1 mole ratio of pyrithione: glucosamine, a 0.2:1weight ratio of sulfur: chitosan, based on one mole sulfur per molepyrithione is expected. The results above for the chitosan-pyrithionecomplex indicate measured sulfur: chitosan ratios of 0.027 initially,falling an order of magnitude after detergent rinsing. Thus, for thechitosan-pyrithione case, chelation efficiency is at best approximately14% of the maximum possible and durability is poor relative to thechitosan-metal or chitosan-zinc pyrithione cases. From the data in TableIV, it might be expected then that the use of chitosan-pyrithionecomplex as an antimicrobial in a sponge product is not acceptable sincethe active ingredient is not durable to common washing conditions.

In the following examples and precursors, cellulose sponges containingchitosan and chitosan complexes as described above were prepared andtested for durability of the complex and anti-microbial effectiveness.

Precursor 3

The cellulose sponge blocks and sample of Precursor 3 were prepared inthe following manner:

Cellulose sponge was prepared by mixing together in a stainless steelkettle 14 kg viscose solution (prepared from hemlock pulp commerciallyavailable from Western Pulp Ltd., Vancouver, British Columbia)containing approximately 10% cellulose and 63.6 kg Glauber's salt withcontinuous stirring. Reinforcing fibers comprising 0.53 kg cellulosefiber of <12 mm staple length (Solka Floc obtained from InternationalFiller, Tonawanda, N.Y.) and 0.3 kg rayon fiber, 12 mm staple length(obtained from Minifiber, Co., Johnson City, Tenn.) were added to thekettle (a Type 21Z universal sigma blade mixer sold by WinkworthMachinery, United Kingdom). After all ingredients had been added to thekettle, mixing was continued for an additional 30 minutes. Total volumeof the mixture was approximately 76 l.

The mixture was poured into a rectangular fiberglass tank approximately51 cm×51 cm×46 cm containing a drain spigot at the bottom and two steelelectrodes, one at each opposing end of the tank. The electrodes wereconnected to an alternating current power source and sufficient voltagewas applied to the electrodes to cause the mixture to heat to atemperature above 95° C. for of total time of 30 minutes at 105 voltsAC, thus regenerating cellulose from the viscose mixture. The saltsolution was liberated from the mixture during the process ofregeneration was drained from the bottom of the tank and the cellulosesponge block was removed from the tank and rinsed with hot tap water(approximately 55° C.) for 30 minutes. The blocks were subsequentlyrinsed with water, 0.3% bleach solution (sodium hypochlorite) and wateragain. Thereafter, after having been thoroughly squeezed between rinsesto remove excess rinsate a sponge block having the dimensions ofapproximately 50 cm×50 cm×30 cm was obtained. The sponge block was cutinto sponge samples approximately 9 cm×16 cm×3 cm.

Comparative Example A

A sponge block was prepared according to the procedure described inPrecursor 3 except that after the final water rinse the block wassqueezed to remove excess water and then immersed in a solutioncontaining 0.3% alkyl benzyl dimethyl ammonium chlorides (ADBAC), acommonly used quaternary ammonium chloride disinfectant containing analkyl group distribution of 40% C₁₂, 50% C₁₄, 10% C₁₆ and sold as MAQUATMC1412 (commercially available from Mason Chemical, Arlington Hts.,Ill.). The sponge block was then squeezed to remove excess rinsate, andcut into sponge samples.

Precursors 4-9

The sponge blocks of Precursors 4 to 9 were prepared according to themethod of Precursor 3 except that chitosan-metal complexes were added tothe viscose mixture prior to the regeneration. The chitosan-metalcomplexes were prepared according to the procedure described inPrecursors 1 using amounts of chitosan and metal sulfate as shown inTable V. The metal analysis results shown in Table V indicate that theamount of zinc relative to the chitosan is approximately 10% based onthe weight of chitosan present, a result which is equivalent to thatshown in Precursors 1 for chitosan-metal filter cake samples. Thisconfirms that metal content survives the viscose regeneration processand subsequent water and bleach rinsing. A background level of 20-30 ppmzinc is present for all samples tested. Similar results were obtainedfor the sponge block containing chitosan and copper.

                                      TABLE V                                     __________________________________________________________________________    Sponge Blocks containing Chitosan complexes.                                                           % Chitosan                                                              % Chitosan                                                                          based on                                                                             Metal                                                            Cellulose                                                                           final  Analysis                                      Chitosan       MSO.sub.4                                                                         Cellulose                                                                           regenerated                                                                          (ppm)                                         Precursor                                                                           (kg)(1)                                                                            Metal                                                                             (kg)(2)                                                                           (3)   sponge solids                                                                        Zn(4)                                                                             Cu                                        __________________________________________________________________________    4     0.0135                                                                             --  0   1     0.62   23  9                                         5     0.1352                                                                             --  0   10    5.8    19  9                                         6     0.068                                                                              Cu  0.068                                                                             5     3.0    <20 600                                       7     0.0135                                                                             Zn  0.0151                                                                            1     0.62   539 <10                                       8     0.068                                                                              Zn  0.0755                                                                            5     3.0    3038                                                                              <10                                       9     0.1352                                                                             Zn  0.151                                                                             10    5.8    --  --                                        CA    0    --  0   0     0      26  14                                        __________________________________________________________________________     Notes                                                                         (1) Chitosan VNS461 was used for all Precursors and Examples in this tabl     except for Presursor 6 which used VNS457.                                     (2) MSO.sub.4 = CuSO.sub.4.5H.sub.2 O or ZnSO.sub.4 7H.sub.2 O as             indicated by the metal.                                                       (3) Calculated percent chitosan based on cellulose content of viscose         solution.                                                                     (4) Metal content of sponge samples was determined by ICP analysis.      

Precursors 10, 11, and 12 and Comparative Example B

Sponge samples of Precursors 10 to 12 and Comparative Example B wereprepared according to methods described in Precursors 6, 7, and 8 andControl Example A respectively. The corresponding samples were dippedinto running 65° C. tap water for approximately two seconds and thenpassed through a two-roll, zero clearance wringer having rubber rolls of20-25 shore gauge A hardness. This process of saturating and wringingthe sponge samples was repeated a total of ten times per sample. Theresidual zinc or copper content of each sample and obtained by ICPanalysis is shown in Table VI. Comparison of metal contents in Table VIwith corresponding Precursors in Table V indicate a substantialproportion of metal is bound by the chitosan even after multiplewashings with water.

Comparative Example 13

Sponge samples containing 1% chitosan and no added zinc were preparedaccording to the procedure outlined in Precursor 4. These samples weretreated with pyrithione acid (PA) as follows: 3.2 g sodium pyrithionesolution (40%) were diluted with 1758 g of water. To this solution wasadded 42 g. of 1% sulfuric acid solution by slow addition with goodstirring yielding approximately 1800 ml of dilute pyrithione acid (PA).

The sponges were then treated by a treatment hereinafter referred as the"potentiation treatment" described as follows: Sponges from preparedaccording to Precursor 4 were placed in a plastic bag, the PA solutionwas added and the bag was sealed. The sponges were squeezed severaltimes and allowed to rest approximately five minutes. Thesqueeze-and-rest potentiation procedure was repeated for a total ofapproximately 30 minutes.

The sponge samples were then subjected to the ten rinse-and-wring cyclesdescribed in Precursors 10-12. The test results are reported in TableVI.

Example 14

Sponge samples containing 10% chitosan and no added zinc were preparedaccording to method described in Precursor 5. These samples were giventhe potentiation treatment as described in Comparative Example 13 exceptthat the PA solution was prepared from 29.9 g 40% sodium pyrithionesolution, 1385 g dilution water and 393 g. of 1% sulfuric acid. Thesamples were then subjected to the rinse-and-wring cycles described inPrecursors 10-12. The test results are reported in Table VI.

Example 15

Sponge samples containing 1% chitosan and added zinc were preparedaccording to the method outlined in Precursor 7. The samples weretreated with sodium pyrithione solution (NAP) as described inComparative Example 13. The NaP solution comprised 6.3 g 40% sodiumpyrithione solution diluted with 1797 g deionized water. The sampleswere then subjected to the ten rinse-and wring cycles described inPrecursors 10-12. The test results are reported in Table VI.

Example 16

Sponge samples containing 10% chitosan and added zinc were preparedaccording to the method outlined in Precursor 9. These samples weretreated with NaP instead of PA as described in Example 15 except thatthe NaP comprised 59.8 g 40% sodium pyrithione solution and 1744 gdeionized water. The samples were then subjected to the tenrinse-and-wring cycles described in Precursors 10-12. The test resultsare reported in Table VI.

Example 17

Sponge samples prepared according to the procedure described inPrecursor 4 contained 1% chitosan but with no added zinc added to theviscose mixture were treated first with zinc sulfate solution and thenwith NaP as follows: using the plastic bag procedure described inComparative 13, six sponge samples from Precursor 4 were placed in aplastic bag containing approximately 1800 ml of zinc sulfate solutioncomprising 2.4 g ZnSO₄ ·7H₂ O dissolved in 1800 ml DI water. After thesesponge samples were squeezed and soaked for 30 minutes according to thepotentiation treatment procedure, they were wrung once in thezero-clearance wringer. The sponge samples were then treated with NaP asdescribed in Example 15 again using the potentiation treatmentprocedure. The samples were then subjected to the ten rinse and wringcycles described in Precursors 10 to 12.

Example 18

Sponge samples prepared according to the method of Precursor 5 contained10% chitosan but with no added zinc were treated first with zinc sulfatesolution and then with NaP as follows: using the plastic bagpotentiation procedure described in Comparative Example 13, six spongesamples from Precursor 5 were placed in the plastic bag containingapproximately 1800 ml of zinc sulfate solution comprising 23.1 g ZnSO₄·7H₂ O dissolved in 1800 ml DI water. After these sponge samples weresqueezed and soaked for approximately 30 minutes according to thepotentiation procedure, they were wrung once through the zero-clearancewringer. The sponge samples were then treated with NaP as described inExample 16 again using the potentiation procedure. The samples were thensubjected to the ten rinse and wring cycles described in Precursors10-12.

Comparative Examples 19, 20

Sponge samples of Comparative Examples 19 and 20 were prepared accordingto the procedures described in Precursors 4 and 5 respectively. Thesamples were then rinsed under tap water repeatedly, sufficientlysqueezed to remove excess water, and then treated with copper sulfatesolution by the plastic bag potentiation technique described inComparative Example 13.

Comparative Example 19 sponge samples were prepared by treating rinsedprecursor sponge samples prepared in accordance with the procedure ofPrecursor 4 with copper sulfate solution which was prepared bydissolving 2.1 g CuSO₄ ·5H₂ O in about 1800 ml DI water. These spongesamples were then subjected to ten rinse and wring cycles described inPrecursors 10-12.

Similarly, Comparative Example 20 sponge samples were prepared bytreating rinsed precursor sponge samples prepared in accordance with theprocedure of Precursor 5 with a copper sulfate solution prepared from 20g CuSO₄ ·5H₂ O and 1800 ml DI. These samples also received the tenrinse-and-wring cycles.

                  TABLE VI                                                        ______________________________________                                        Metal Content* of Treated Sponge Samples                                      Example/                                                                      Precursor   Treatment   Cu(ppm)    Zn(ppm)                                    ______________________________________                                        B           Water       <10        27                                         10          Water       622        8                                          11          Water       <20        452                                        12          Water       13         5354                                       13          PA          <4         4                                          14          PA          4          4                                          15          NaP         <4         340                                        16          NaP         7          4627                                       17          Zn, NaP     1          300                                        18          Zn, NaP     5          4014                                       19          Cu          229        9                                          20          Cu          2530       9                                          ______________________________________                                         *By ICP analysis                                                         

Comparison of Precursors 11 and 12 and Examples 15, 16, 17, and 18 andComparative Examples 19 and 20 indicate that the metal content generallyis near 10% of and is proportional to chitosan content which isconsistent with the findings in Precursor 1. It is also interesting thatthe zinc content of the sponges is within 10% of the zinc content forthose samples in which zinc was introduced prior to regeneration(Examples 15, 16) or after regeneration (Examples 17, 18).

Antimicrobial Activity Test Procedure

Sponge samples were tested for anti-microbial activity by measuringtheir "preservative effectiveness" (PE) as described below. Spongesamples were cut into strips approximately 9 cm×3 cm×1.5 cm. Each stripwas inoculated with 10⁶ colony-forming units (CFU) of bacteria or 10⁵CFU of fungi by allowing 3.0 ml inoculum to be absorbed by the spongestrip. Bacteria were chosen from the set of Pseudomonas aeruginosa (ATCC15442), Staphylococcus aureus (ATCC 6538), and Eschericha coli (ATCC8739). The fungi were chosen from the set of Aspergillus niger (ATCC9642), Candida albicans (ATTC 10231), and Chaetomium globosum (ATCC6205). The inoculated strips were placed into individual, sterile,sealable plastic bags and stored at room temperature. An uninoculatedsample was similarly stored as the control sample. Bacteria inoculateswere stored for seven days while fungi and uninoculated samples werestored for 14 days.

Microbial growth per sponge strip was determined in the followingmanner. Each sponge strip was placed in a "stomacher" laboratory blender(Tekmar, Ltd, England; obtainable from Baxter Scientific Co, catalognumber H3493-2), 100 ml of letheen broth was added, and the sample wasprocessed one minute. Aliquots of the liquid were withdrawn and used toculture pour plates containing Letheen agar. The plates were incubated48 hr at 32° C. for bacterial and 7 days at 25° C. for fungal anduninoculated samples. Using standard plate counting techniques, thenumber of CFU relative to the original inoculum was determined.

The difference between the inoculum count and the sample was thendetermined. The test results are reported as the logarithm of thisdifference. These treatments were then evaluated according to thestandard described in U.S. Pharmacopeia, Volume XXII, 1990. A logreduction of 3.0 or greater (corresponding to reduction in microbialpopulation of 99.9%) is considered "high" activity. A log reductionbetween 1.0 and 3.0 might be considered "moderate activity." Results forPE testing of sponge samples described above are shown in Table VII.

Sponge sample strips from Precursors 10-12 and Examples 13-20 weretested both for immediate activity as well as longer term antimicrobialactivity. Immediate activity was determined by analyzing samples within30 minutes of inoculation. The results are indicated in Tables VII andVIII as "Immed." For longer term activity, samples inoculated withbacteria were analyzed after seven days storage whereas samplesinoculated with fungi were analyzed after fourteen days storage. Theseresults are indicated in Table VII and VIII as "Fin."

                                      TABLE VII                                   __________________________________________________________________________    Antimicrobial Activity of Sponge Samples (1)                                  Example/           Ps.                                                        Comparative        aeruginosa                                                                           S. aureus                                                                            E. coli A. niger                                                                             C. albicans                                                                          C. globosom            Examples                                                                              Metal                                                                             Treatment (2)                                                                        Immed                                                                             Fin                                                                              Immed                                                                             Fin                                                                              Immed                                                                             Fin Immed                                                                             Fin                                                                              Immed                                                                             Fin                                                                              Immed                                                                             Fin                __________________________________________________________________________    B (3)   --  --     0.00                                                                              6.62                                                                             0.00                                                                              6.82                                                                             3.49                                                                              6.98                                                                              2.54                                                                              4.93                                                                             5.12                                                                              5.12                                                                             3.30                                                                              3.48               10      Cu  --     0.00                                                                              0.00                                                                             0.00                                                                              6.82                                                                             0.00                                                                              -0.06                                                                             2.31                                                                              0.00                                                                             0.00                                                                              0.89                                                                             2.6 -0.03              11      Zn  --     0.00                                                                              0.00                                                                             -0.16                                                                             6.82                                                                             0.00                                                                              1.22                                                                              2.41                                                                              2.42                                                                             0.00                                                                              -0.89                                                                            2.88                                                                              0.53               12      Zn  --     0.00                                                                              1.12                                                                             0.00                                                                              6.52                                                                             1.03                                                                              7.07                                                                              2.37                                                                              2.52                                                                             0.64                                                                              2.10                                                                             2.98                                                                              3.35               13      --  PA     0.00                                                                              1.23                                                                             0.00                                                                              1.48                                                                             0.00                                                                              1.48                                                                              3.24                                                                              5.8                                                                              0.00                                                                              6.86                                                                             3.04                                                                              3.87               14      --  PA     0.00                                                                              2.23                                                                             0.00                                                                              3.92                                                                             0.00                                                                              3.92                                                                              3.33                                                                              5.00                                                                             0.00                                                                              4.98                                                                             3.38                                                                              4.17               15      Zn  NaP    0.00                                                                              1.82                                                                             0.00                                                                              7.08                                                                             0.00                                                                              7.08                                                                              3.46                                                                              7.41                                                                             0.00                                                                              6.86                                                                             2.70                                                                              3.23               16      Zn  NaP    0.00                                                                              2.56                                                                             0.00                                                                              6.52                                                                             1.03                                                                              7.07                                                                              2.42                                                                              6.19                                                                             0.65                                                                              6.91                                                                             3.56                                                                              4.26               17      Zn  NaP    0.00                                                                              2.44                                                                             0.00                                                                              6.52                                                                             1.03                                                                              7.07                                                                              2.48                                                                              7.41                                                                             0.72                                                                              6.91                                                                             3.21                                                                              5.94               18      Zn  NaP    0.00                                                                              1.15                                                                             -0.17                                                                             6.52                                                                             1.03                                                                              7.07                                                                              2.45                                                                              2.99                                                                             1.01                                                                              5.21                                                                             5.95                                                                              5.95               19      Cu  --     0.00                                                                              0.00                                                                             0.00                                                                              6.82                                                                             0.00                                                                              0.85                                                                              2.34                                                                              2.57                                                                             0.00                                                                              -0.90                                                                            3.00                                                                              3.48               20      Cu  --     0.00                                                                              5.76                                                                             0.00                                                                              5.47                                                                             0.00                                                                              5.47                                                                              3.27                                                                              4.70                                                                             0.00                                                                              6.86                                                                             2.65                                                                              3.90               __________________________________________________________________________     (1) Expressed as logarithm of inoculum population reduction.                  (2) Other than water rinses.                                                  (3) Originally treated with quaternary disinfected as Control Example A. 

ANTIMICROBIAL ACTIVITY OF TREATED SPONGE SAMPLES AFTER LAUNDERINGExample 21

Twelve sponge samples containing chitosan and zinc were preparedaccording to the method described in Precursor 7. These samples weretreated by the potentiation procedure as described in ComparativeExample 13 with NaP solution comprising 63.1 g 40% NaP diluted with 3544g DI water. The sponge samples were dipped into warm tap water andsqueezed by hand for a total of ten repetitions. The samples were thensubjected to the ten rinse and wring cycles as described in Precursors10-12. Four of these treated sponge samples were then laundered in ahousehold washing machine using a medium water level (approximately 15gal), hot wash, warm rinse, and "normal" agitation speed.

Approximately 68 g of "Tide" powdered laundry detergent was added to thewash cycle. These sponges were then subjected to a final launderingcycle as just described except no detergent was added. This final cyclewas intended to ensure removal of laundry detergent from the sponges.These samples are indicated as "2X" laundry cycles in Table VIII whereresults for PE testing are presented.

A second set of four sponge samples prepared above was laundered asdescribed above except that four laundry cycles (including detergent)were completed followed by a fifth cycle without detergent. Thesesamples are noted in Table VIII as "5X" laundered.

The third set of four sponges prepared above was not laundered and aredesignated as "OX" laundered.

Comparative Example 22

Twelve sponge samples containing chitosan and no added metal wereprepared according to the procedure described in Precursor 5. Thesesamples were treated as described in Comparative Example 13 with PAsolution comprising 59.8 g 40% NaP solution diluted with 3056 g DI waterto which was added with stirring a solution comprising 7.95 g 98%sulfuric acid diluted with 500 g DI water. These sponges were thenrinsed and laundered as described in Example 21. These sponge sampleswere then subjected to PE testing and the results were reported in TableVIII.

Comparative Example 23

Sponge samples were prepared as described in Comparative Example A.These samples were subjected to the rinse and laundering procedures ofExample 21. These samples contain the quaternary disinfectant from theirpreparation. These samples were then subjected to PE testing and theresults are shown in Table VIII.

                                      TABLE VIII                                  __________________________________________________________________________    ANTIMICROBIAL ACTIVITY OF LAUNDERED SPONGES                                             Laundered                                                                           Ps. aeruginosa                                                                        S. aureus                                                                           E. coli                                                                             A. niger                                                                            C. albicans                                                                         C. globosom                   Ex.                                                                              Treatment                                                                            Cycles                                                                              Imm Fin Imm                                                                              Fin                                                                              Imm                                                                              Fin                                                                              Imm                                                                              Fin                                                                              Imm                                                                              Fin                                                                              Imm                                                                              Fin                        __________________________________________________________________________    21 Zn, NaP                                                                              0X    0.58                                                                              6.61                                                                              0.36                                                                             6.57                                                                             0.78                                                                             7.18                                                                             1.53                                                                             6.33                                                                             0.00                                                                             6.23                                                                             6.23                                                                             5.38                                 2X    0.72                                                                              6.14                                                                              0.50                                                                             6.95                                                                             0.72                                                                             6.14                                                                             1.47                                                                             6.30                                                                             0.58                                                                             6.85                                                                             2.51                                                                             4.95                                 5X    0.59                                                                              -0.04                                                                             0.32                                                                             -0.51                                                                            0.87                                                                             0.14                                                                             1.59                                                                             1.60                                                                             -0.12                                                                            3.34                                                                             3.12                                                                             5.38                       22 PA     0X    0.57                                                                              0.59                                                                              0.20                                                                             6.57                                                                             0.74                                                                             2.07                                                                             1.62                                                                             6.33                                                                             -0.01                                                                            6.23                                                                             2.97                                                                             5.38                                 2X    0.84                                                                              *   0.54                                                                             *  0.80                                                                             *  1.58                                                                             *  0.61                                                                             *  4.52                                                                             *                                    5X    0.56                                                                              *   0.38                                                                             *  0.71                                                                             *  1.55                                                                             *  -0.12                                                                            *  3.10                                                                             *                          23 Quaternary                                                                           0X    0.00                                                                              6.62                                                                              6.95                                                                             6.95                                                                             6.69                                                                             7.41                                                                             1.60                                                                             2.95                                                                             1.64                                                                             6.85                                                                             3.14                                                                             3.57                          Disinfectant                                                                         2X    0.00                                                                              0.00                                                                              0.39                                                                             0.22                                                                             0.81                                                                             0.49                                                                             1.44                                                                             2.08                                                                             0.51                                                                             6.85                                                                             2.45                                                                             3.52                       __________________________________________________________________________     *Sample plates not readable; contaminated by noninoculate organisms.     

The data in Table VIII indicate lack of durability towards laundering,and therefore towards normal household usage, of the quaternarydisinfectant and pyrithione acid (PA) sponge treatments. Efficacy of thequaternary disinfectant against bacteria was significantly diminished bylaundering. PA treatment of the sponge samples prior to laundering washighly effective against all but two bacteria. However, afterlaundering, the sponge samples allowed a native organism to flourishthus eliminating the possibility of reading the presence of inoculatedorganisms. That all sponge samples in Table VIII were rinsed andlaundered similarly and separately suggest that the PA treated spongewas particularly susceptible to biological attack and perhaps more sothan compared to the sponge without chitosan having been treated withwater leachable quaternary ammonium disinfectant. Chitosan itself isbiodegradable and, in the absence of an antimicrobial agent, eitherinherent or added, becomes a suitable food source for organisms.

Pyrithione acid sponge samples of Comparative Example 22 containedchitosan at 10% of the viscose cellulose whereas the zinc pyrithionesponges of Example 21 contained 1% chitosan. In many cases at 0Xlaundering, these were equally effective against the inoculates.However, for two important gram-negative species, E. coli and Ps.aeruginosa, the zinc pyrithione treatment was significantly superior tothe PA treatment of chitosan-containing sponges. Also, at 10%,especially in dry sponges, the chitosan tends to be friable and wasnoticeable in the sponge as particulate matter. Aesthetically, thepresence of fragmentable particulates in the sponge is unpleasant, andtherefore the zinc pyrithione treatment at 1% chitosan is more to bepreferred. This treatment also results in high activity (>3 log unitsreduction) against all tested organisms after two laundry cycles andeven after five laundry cycles shows excellent activity against C.albicans and C. globosum. The latter organism has cellulolytic activityand may therefore contribute, if allowed to grow in the sponge, to thephysical degradation of the sponge structure over time.

Comparative Examples 24A-D and Examples 24E-R.

The following examples and comparative examples demonstrate thedurability of the chitosan-zinc-pyrithione complex in relation toviscose regeneration and more specifically in relation to the typicalsponge manufacturing process. The untreated chitosan VNS-461 will bereferred to as Comparative Example 24A. In a 300 cc flask, 15.6 g ofZnSO₄ ·7H₂ O was dissolved into 162 g of DI water. After completedissolution of the zinc salt, 8.5 g of VNS-461 grade chitosan was addedand stirred 1 hr. The resulting zinc cake was filtered by vacuum andrinsed with 1000 cc DI water on the filter. One seventeenth of this cakewas removed and dried at 50C for 3 hours, and will be referred to asComparative Example 24B. The remaining zinc cake was added to a roundbottom flask with 500 cc of 1.5% NaOH in DI water and boiled for 1 hrwith total reflux. The cake was drained, filtered by vacuum, and rinsedwith 2000 cc of DI water on the filter. One sixteenth of this cake wasremoved and dried at 50C for 3 hours, and will be referred to asComparative Example 24C. The remaining cake was then added to a flaskwith 3000 cc of 0.3% bleach and stirred for 30 minutes. This cake wasthen filtered by vacuum and rinsed with 2000 cc DI water. One fifteenthof this cake was removed and dried at 50C for 3 hours, and will bereferred to as sample 24D. The remaining cake was then potentiated withpyrithione by adding the cake to a flask with 33.2 g sodium pyrithione(NAP) solution (40%) and 133 g DI water. This slurry was stirred for 30minutes, and then filtered by vacuum, and rinsed with 1000 cc DI on thefilter. The cake was then split into equal 14ths, one of which was driedat 50C for 3 hours and will be referred to as sample 24E. The remainingequal 13 splits were rinsed on the vacuum filter with varying solutionsand quantities as described in Table IX, and dried at 50C for 3 hours.Table IX also shows resultant Zn and S analysis which was done by ICPusing nitric acid digestion of the dried samples.

                                      TABLE IX                                    __________________________________________________________________________    Example 24 sample descriptions.                                               Original                                                                      Cake Treatment                                                                           Rinse          Zn  S    Example                                    __________________________________________________________________________    VNS-461                                                                            NONE  NONE           3. ppm                                                                            ≦5. ppm                                                                     C24A                                       C24A ZnSO.sub.4                                                                          1000 cc DI     9.9%**                                                                            5.1% C24B                                       C24B NaOH  2000 cc DI     11.1%                                                                             37. ppm                                                                            C24C                                       C24C BLEACH                                                                              2000 cc DI     12.1%                                                                             ≦5. ppm                                                                     C24D                                       C24D NaP   1000 cc DI     8.8%                                                                              9.2% 24E                                        24E  NONE  1000 cc DI     9.2%                                                                              9.6% 24F                                        24E  NONE  2000 cc Di     9.0%                                                                              9.6% 24G                                        24E  NONE  4000 cc DI     9.5%                                                                              10.1%                                                                              24H                                        24E  NONE  1000 cc 20C tap water                                                                        8.8%                                                                              9.3% 24I                                        24E  NONE  2000 cc 20C tap water                                                                        9.2%                                                                              9.6% 24J                                        24E  NONE  4000 cc 20C tap water                                                                        9.0%                                                                              9.4% 24K                                        24E  NONE  1000 cc 55C tap water                                                                        9.7%                                                                              10.1%                                                                              24L                                        24E  NONE  2000 cc 55C tap water                                                                        8.8%                                                                              9.1% 24M                                        24E  NONE  4000 cc 55C tap water                                                                        9.8%                                                                              10.3%                                                                              24N                                        24E  NONE  1000 cc 1.6% Spic and Span* +                                                                9.7%                                                                              10.3%                                                                              24O                                                   1000 cc DI                                                         24E  NONE  1000 cc 0.5% bleach* +                                                                       10.7%                                                                             8.5% 24P                                                   1000 cc DI                                                         24E  NONE  1000 cc 1% Comet* +                                                                          9.8%                                                                              10.3%                                                                              24Q                                                   1000 cc DI                                                         24E  NONE  1000 cc 1% Dawn* +                                                                           10.2%                                                                             10.8%                                                                              24R                                                   1000 cc DI                                                         __________________________________________________________________________     *Comet Cleanser, Dawn Dishwashing Liquid, and Spic and Span are all           commercially available from Proctor and Gamble, Cincinnati OH. Clorox         bleach is available from Clorox, Oakland CA.                                  **% = percentage by weight.                                              

The data in Table IX indicate that zinc pyrithione is durable to thesecommon rinses.

Comparative Examples 25 A-D and Example 25E-H

Zinc pyrithione is susceptible to discoloration due to presence of iron,as a result of chelation of free pyrithione with available iron. Out ofconcern that the chitosan-zinc-pyrithione complex might be susceptibleto loss of pyrithione when subjected to what would be considered highiron (10 ppm) or Ca and Mg ion (300 ppm) concentrations in hard watersituations, the following experiment was conducted. In a 300 cc flask,6.4 g of ZnSO₄ ·7H₂ O was dissolved into 67 g of DI water. Aftercomplete dissolution of the zinc salt, 3.5 g of VNS-461 grade chitosanwas added and stirred 1 hr. The untreated chitosan will be referred toas Comparative Example 25A. The resulting zinc cake was filtered byvacuum and rinsed with 1000 cc DI water on the filter. One seventh ofthis cake was removed and dried at 50C for three hours, and will bereferred to as Comparative Example 25B. The remaining zinc cake wasadded to a round bottom flask with 500 cc of 1.5% NaOH in DI water andboiled for 1 hr with total reflux. The cake was drained, filtered byvacuum, and rinsed with 2000 cc of DI water on the filter. One sixth ofthis cake was removed and dried at 50C for 3 hours, and will be referredto as Comparative Example 25C. The remaining cake was then added to aflask with 3000 cc of 0.3% bleach and stirred for 30 minutes. This cakewas then filtered by vacuum and rinsed with 2000 cc DI. One fifth ofthis cake was removed and dried at 50C for 3 hours, and will be referredto as Comparative Example 25D. The remaining cake was then potentiatedwith pyrithione by adding the cake to a flask with 9.5 g sodiumpyrithione (NAP) solution (40%) and 38 g DI water. This slurry wasstirred for 30 minutes, and then filtered by vacuum, and rinsed with1000 cc DI on the filter. The cake was then split into equal fourths,one of which was dried at 50C for 3 hours and will be referred to asComparative Example 25E. The remaining equal three splits were rinsed onthe vacuum filter with 4000 cc hard water solutions as described inTable X, and dried at 50C for 3 hours. Table X also shows resultant Znand S analysis which was done by ICP using nitric acid digestion of thedried samples as described in Precursors 1a-g.

                                      TABLE X                                     __________________________________________________________________________    Comparative Examples 25A-D and Example 25E-H sample descriptions.             Original                                                                      Cake Treatment                                                                           Rinse         Zn   S    Example                                    __________________________________________________________________________    VNS-461                                                                            NONE  NONE          18. ppm                                                                            26. ppm                                                                            25A                                        C25A ZnSO.sub.4                                                                          1000 cc DI    9.7%**                                                                             5.1% 25B                                        C25B NaOH  2000 cc DI    9.6% 39. ppm                                                                            25C                                        C25C BLEACH                                                                              2000 cc DI    10.2%                                                                              65. ppm                                                                            25D                                        C25D NaP   1000 cc DI    7.7% 8.1% 25E                                        25E  NONE  8000 cc 20C DI                                                                              7.1% 7.5% 25F                                        25E  NONE  4000 10 ppm *Fe water +                                                                     6.5% 7.1% 25G                                                   4000 cc Di                                                         25E  NONE  4000 cc 300 ppm *Ca water +                                                                 6.7% 7.2% 25H                                                   4000 cc DI                                                         __________________________________________________________________________     *10 ppm Fe water comprised of 0.2 g FeSO.sub.4 *7H.sub.2 O added to 4000      cc DI; 300 ppm Ca water comprised of 4.4 g CaCl.sub.2.2 2H29 and 4.9 9        MgSO407H20 added to 4000 cc DI.                                               **% = percentage by weight.                                              

The results shown in Table X indicate no significant loss of eithersulfur or zinc, and thus no loss of the pyrithione moiety, for eitherwater, iron or Ca and Mg rinses. The results further indicate thedurability of the active complex.

Comparative Example 26A and Example 26B-F

In a 1000 cc flask, 33 g of ZnSO₄ ·7H₂ O was dissolved into 342 g of DIwater. After complete dissolution of the zinc salt, 18 g of VNS-461grade chitosan was added and stirred 1 hr. The resulting zinc cake wasfiltered by vacuum and rinsed with 1000 cc DI water on the filter.Approximately 4 g of this damp cake was removed and dried at 50C for 3hours, and will be referred to as Comparative Example 26A. The remainingcake was then potentiated with pyrithione by adding the cake to a flaskwith 76 g sodium pyrithione (NAP) solution (40%) and 304 g DI water.This slurry was stirred for 30 minutes, and then filtered by vacuum.Approximately 4 g of this damp cake was removed and dried at 50C forthree hours, and will be referred to as Comparative Example 26B. Thecake was then split into equal sevenths, which were washed separately onthe filter with hot water and two common cleaning solutions as describedin Table XI. The cakes were then dried for 3 hrs at 50C. Thereafter, thecakes were tested for metal content using the ICP analysis described inPrecursors 1a-g.

                                      TABLE XI                                    __________________________________________________________________________    Chitosan-zinc-pyrithione sample descriptions and durability to rinsing.       Original                                                                      Cake Treatment                                                                           Rinse      Zn    S     Example                                     __________________________________________________________________________    VNS-461                                                                            NONE  NONE       ≦25. ppm                                                                     ≦30. ppm                                   VNS-461                                                                            ZnSO.sub.4                                                                          1000 cc DI 9.8%**                                                                              5.1%  26A                                         26A  NaP   NONE       6.8%  9.7%  26B                                         26B  NONE  1000 cc 55C tap water                                                                    8.4%  8.6%  26C                                         26B  NONE  4000 cc 55C tap water                                                                    8.3%  8.4%  26D                                         26B  NONE  1000 cc Spic and Span +                                                                  7.5%  7.5%  26E***                                                 3000 cc DI water                                                   26B  NONE  1000 cc Dawn +                                                                           10.3% 10.9% 26F***                                                 3000 cc DI water                                                   __________________________________________________________________________     *Spic and Span solution comprised of 1.6 wt % Spic and Span in water; Daw     Dishwashing Liquid concentration employed was 1 wt % Dawn in water.           **% = percentage by weight.                                                   ***for samples 26E & 26F, the 3000 cc DI rinse followed the first 1000 cc     rinse with cleansing solutions                                           

These results indicate again the durability of the activechitosan-zinc-pyrithione complex.

Comparative Example 27A and Example 27B-F

Chitosan-zinc-mercaptobenzothiazole complex was prepared and rinsed withcleansing solutions in accordance with Examples 26 except instead ofusing sodium pyrithione for potentiation of the zinc cake, 77 g ofsodium mercaptobenzothiazole (NaMBT) solution (50%) was employed withthe 304 g dilution water (sodium mercaptobenzothiazole is soldcommercially as NACAP from the R.T Vanderbilt Company, Norwalk Conn.).It should be noted that the resultant cake turned bright yellow uponchelation of NaMBT with the chitosan-zinc complex. Sample descriptionand metal analysis results are presented in Table XII, and the resultsshow the comparable durability of this active relative to thechitosan-zinc-pyrithione case.

                  TABLE XII                                                       ______________________________________                                        Chitosan-zinc-mereaptobenzothiazole sample                                    descriptions and durability to rinsing.                                       Origi-                                    Ex-                                 nal   Treat-                              am-                                 Cake  ment     Rinse          Zn    S     ple                                 ______________________________________                                        VNS-  ZnSO.sub.4                                                                             1000 cc DI     10.3% 5.3%  27A                                 461                                                                           C27A  NaMBT    NONE           7.6%  13.9% 27B                                 27B   NONE     1000 cc 55C tap water                                                                        9.7%  11.7% 27C                                 27B   NONE     4000 cc 55C tap water                                                                        10.3% 12.2% 27D                                 27B   NONE     1000 cc Spic and Span +                                                                      9.5%  10.6% 27E                                                3000 cc DI water                                               27B   NONE     1000 cc Dawn + 10.6% 13.0% 27F                                                3000 cc DI water                                               ______________________________________                                    

Comparative Example 28A and Example 28B-F

Chitosan-zinc-dimethyldithiocarbamate complex was prepared and rinsedwith cleansing solutions in accordance with Examples 26 except insteadof using sodium pyrithione for potentiation of the zinc cake, 100 g ofsodium dimethyldithiocarbamate (NaDMDTC) solution (30%) was employedwith the 304 g dilution water (NaDMDTC is sold commercially as Vancide51 from the R.T. Vanderbilt Company, Norwalk Conn.). It should be notedthat the resultant cake became more white relative to the original lighttan colored chitosan cake upon chelation of NaDMDTC with thechitosan-zinc complex. Sample description and metal analysis results arepresented in Table XIII, and the results show the comparable durabilityof this active relative to the chitosan-zinc-pyrithione case.

                  TABLE XIII                                                      ______________________________________                                        Chitosan-zinc-dimethyldithiocarbamate sample                                  descriptions and durability to rinsing.                                                                                 Ex-                                 Original                                  am-                                 Cake   Treatment  Rinse       Zn    S     ple                                 ______________________________________                                        VNS-461                                                                              ZnSO.sub.4 1000 cc DI  10.3% 5.2%  28A                                 28A    NaDMDTC    NONE        7.3%  21.9% 28B                                 28B    NONE       1000 cc 55C tap                                                                           8.3%  18.4% 28C                                                   water                                                       28B    NONE       4000 cc 55C tap                                                                           8.5%  20.2% 28D                                                   water                                                       28B    NONE       1000 cc Spic                                                                              8.1%  18.8% 28E                                                   and Span +                                                                    3000 cc DI                                                                    water                                                       28B    NONE       1000 cc Dawn +                                                                            8.0%  21.0% 28F                                                   3000 cc DI                                                                    water                                                       ______________________________________                                    

Comparative Examples 29

The following example illustrates the advantage of the pre-chelation ofzinc ion followed by potentiation with an antimicrobial agent inimparting durability to the active final chitosan complex. Forcomparison to the chitosan-zinc-potentiator complexes described inExamples 26, 27 and 28, a chitosan-pyrithione complex was prepared. Asodium pyrithione (NAP) solution was prepared by stirring 42 g of NaPsolution (40%) into 1292 g DI water. To this solution was added 272 g ofa 2 wt % sulfuric acid solution comprised of 5.4 g H₂ SO₄ diluted in266.6 g DI water. The acid solution was added slowly with good stirringfor 30 minutes. To this resultant pyrithione acid (PA) solution was thenadded 16 g of VNS-461 chitosan which was allowed to stir 1 hr. Thisslurry was then filtered by vacuum, and 4 g of the damp cake was removedand dried at 50C for 3 hrs. This dried cake will be referred to asComparative Example 29B.(Note: for consistency of numbering nomenclatureno Comparative Example 29A has been designatied). The remaining dampcake corresponding to Comparative Example 29B was then rinsed with waterand cleaning solutions by the procedure described in Example 26. Sampledescription and sulfur analysis results are presented in Table XIV.

                  TABLE XIV                                                       ______________________________________                                        Chitosan-pyrithione sample descriptions                                       and durability to rinsing.                                                    Origi-                                    Ex-                                 nal   Treat-                              am-                                 Cake  ment    Rinse          Zn      S    ple                                 ______________________________________                                        VNS-  PA      NONE           ≦10. ppm                                                                       7.1% 29B                                 29B   NONE    1000 cc 55C tap water                                                                        ≦10. ppm                                                                       6.4% 29C                                 29B   NONE    4000 cc 55C tap water                                                                        ≦10. ppm                                                                       6.2% 29D                                 29B   NONE    1000 cc Spic and Span +                                                                      ≦10. ppm                                                                       2.9% 29B                                               3000 cc DI water                                                29B   NONE    1000 cc Dawn + ≦10. ppm                                                                       7.3% 29F                                               3000 cc DI water                                                ______________________________________                                    

The results shown in Table XIV indicate a significant loss of sulfur andthus pyrithione when the complex was rinsed with Spic and Span cleaningsolution. Thus the chitosan-pyrithione complex is seen to beconsiderably less durable relative to the chitosan-zinc-pyrithionecomplexes of Examples 26, 27 and 28.

Samples 29D, 28D, 27D, and 26D, having all received4000 cc hot waterrinsing, were then subjected to testing for antimicrobial activity usinga Potato Dextrose Agar plate method as were untreated chitosan VNS-461,and samples 26A, 27A, and 28A which are the unpotentiated chitosan-zinccomplexes. The procedure for this activity test is described below. Moldgrowth agar, sold commercially as Potato Dextrose Agar from BaltimoreBiological Laboratories, was supplied as a dehydrated powder. This wasrehydrated for use in cultivation of molds as per directions supplied bythe manufacturer. After rehydration, the medium was sterilized byautoclaving at 121C and 15 psi for 15 minutes. To test the activity ofthe chitosan samples, 0.225 g of each chitosan sample was added to thebottom of sterile petri dishes, to which 22.5 cc molten Potato DextroseAgar (PDA) was added. This resulted in approximately a 1 percent byweight amount of sample in the PDA active mixture. The chitosan andtreated chitosan samples were dispersed into the agar by swirling of thesolution via manual circular rotation of the plates. These suspensionswere then allowed to harden in the plates by standing covered at roomtemperature on the benchtop. A control plate was prepared with PDA byitself. All plates were prepared in triplicates from the same sampleexcept for the chitosan-zinc complex plates for which a single plate wasprepared from samples 26A, 27A, and 28A. The plates were then inoculatedwith mold spores using the ASTM G21-90. The molds used included thefollowing: Aspergillus niger, Gliocladium virens, Aureobasidiumpullulans, Chaetomium globosum, and Penicillium funiculosum. The sporesof these molds were dispersed into a phosphate buffer broth in equalnumbers to a final mold concentration of 10⁶ spores/cc. The spores werethen delivered to the prepared plates via a compressed air-drivennebulizer. The spraying continued until the plates were damp. The plateswere then incubated at 28C and 95% relative humidity, and were checkeddaily for mold growth. Results are shown in Table XV, where the day ofobserved growth is recorded.

The results in Table XV show that the chitosan-zinc-potentiatorcomplexes of either pyrithione, mercaptobenzothiazole, ordimethyldithiocarbamate resist mold growth beyond the period of the testwhereas all other samples including chitosan-zinc andchitosan-pyrithione fail within this period.

                  TABLE XV                                                        ______________________________________                                        Potato Dextrose Agar activity test of                                         chitosan complez actives.                                                                                  Days                                                                          incubated                                                                     until                                                                         growth                                                                        (failure)                                        Example      Description     observed                                         ______________________________________                                        PDA          control agar plate                                                                            1                                                VNS-461      untreated chitosan                                                                            1                                                26A, 27A, 28A                                                                              chitosan-zinc complex                                                                         1                                                26D          chitosan-zinc-pyrithione                                                                      >12                                              27D          chitosan-zinc-  >12                                                           mercaptobenzothiazole                                            28D          chitosan-zinc-  >12                                                           dimethyldithiocarbamate                                          29D          chitosan-pyrithione                                                                           4                                                ______________________________________                                    

In the next three examples and comparative examples, the utility of thechitosan-zinc-pyrithione complex in the preparation of cellulose spongewith durable antimicrobial activity is further demonstrated.

Comparative Examples 30A-D

Commercially available white (unpigmented) cellulose sponges wereobtained from the O-Cel-O company, Tonawanda, N.Y. Three sponges hadbeen rinsed with the quaternary ammonium solution as described inComparative Example A, and had the same dimension as sponge samplesdescribed in Precursor 3. Comparative Example 30A comprised three suchsponges. Three other sponges were taken and further rinsed in thefollowing fashion. Hot 55C tap water was run over the spongesindividually until the sponges were satured after which the sponges werewrung out in a zero clearance wringer having rubber rolls with 20-25shore gage A hardness. This saturation and wringing procedure wasrepeated a total of 20 times. The sponge samples were then sealed inplastic bags, and will be referred to as Comparative Examples 30B. Threeadditional sponges were taken and given the same 20 repetitionsaturation and rinsing procedure as for Comparative Examples 30B, and inaddition were dipped in a large beaker with 55C water flowing into it atmaximum flow rate from the house supply, and wrung out in the zeroclearance wringer. This procedure was repeated a total of 30 times toyield sponges which had been wrung out in the zero clearance wringer atotal of 50 repetitions. These sponge samples were also sealed in bagsand will be referred to as Comparative Examples 30C. Three additionalsponges were selected and were rinsed in identical fashion asComparative Examples 30C except an additional 50 repetitions of dippingin the hot water and wringing in the zero clearance wringer were made.These sponges therefore received a total of 100 repetitions of wringingin the zero clearance wringer. These sponge samples were then sealed inplastic bags and will be referred to as Comparative Examples 30D.

Examples 31A-D

A sponge block was prepared in accordance with procedures described inPrecursor 7 having added zinc at 1% relative to the viscose cellulosepresent. Sponge samples were cut from this block as described inPrecursor 3. Twelve of these sponge samples were placed in a plastic bagand were potentiated with sodium pyrithione using the potentiationtreatment described in Comparative Example 13. This was accomplished byadding a dilute sodium pyrithione solution to the bag containing thesponges; the solution being comprised of 53 g of sodium pyrithionesolution (40%) and 2827 g distilled water. These sponges received the 30minute squeeze and rest procedure described in Comparative Example 13 inorder to uniformly distribute the active throughout the sponge and toallow for chelation of the chitosan-zinc complex within the sponge. Itwas noted that immediately upon addition of the NaP solution to the bagof sponges, a purplish discoloration of the treatment solution occurred.This is believed to be due to reaction of sodium pyrithione withavailable iron within the sponge, which was measured to be 60 to 100 ppmin the sponge. It is well known in the art that sodium pyrithione andzinc pyrithione compounds exhibit this discoloration phenomena whencontaminated with excess levels of iron, and that only a few ppm ironwill show strong discoloration. This discoloration was noted todisappear after approximately ten minutes, presumably due to thepreferential chelation of pyrithione with zinc in the sponge. Spongesamples were then wrung out by hand to remove excess solution. Threesponges samples were then sealed in a plastic bag and will be referredto as Example 31A. Sponge samples were rinsed in accordance with the 20,50, and 100 repetition rinsing and wringing procedure described inComparative Example 30, and will be referred to as Examples 31B, 31C,and 31D, respectively.

Examples 32A-D

In view of the transient discoloration observed in Example 31 due topyrithione interaction with iron inherent in the sponge, sponge samplesof Example 32 were prepared employing zinc oxide (ZnO) to prevent irondiscoloration in pyrithione applications. A sponge block was preparedand subsequent cut sponge samples were treated and rinsed in accordancewith the procedure described in Example 31, except that 11 g of ZnOdispersion (50%) was added to the viscose mixture prior to regenerationof the sponge block. The ZnO dispersion was obtained from the Penn ColorCorporation, Doylestown Pa. When cut sponges from this block wereinitially wet with the sodium pyrithione solution as described inExample 31, no discoloration of the treatment solution or the sponge wasobserved. The sponge retained its original white appearance throughoutthe potentiation procedure. The treatment solution also did not becomepurplish throughout the treatment. The sponges were wrung out by handand were sealed in plastic bags after receiving no rinsing, and 20, 50,and 100 rinse treatments described in Example 31. These sponge sampleswill be referred to as Examples 32A, and 32B, 32C, and 32D,respectively.

Sponges from this example and including samples from Comparative Example30 and Example 31 were then subjected to the PE antimicrobial test foractivity against Pseudomonas aeruginosa, as described earlier. Resultsfor this testing are presented in Table XVI, and show the superiordurability of activity for the chitosan-zinc-pyrithione sponges comparedto the similarly treated commercially availables samples, ComparativeExamples 30A, 30B, 30C and 30D.

In addition to the PE antimicrobial testing, sponges from this exampleand samples from Comparative Example 30 and Example 31 were thensubjected to a test for activity against fungal growth. These spongeswere tested in accordance with ASTM Method G21-90 for mold resistance.Two sets of mold were used; one set being the standard organismsdesignated by the ASTM method and the other set being eight molds whichhad been isolated from used sponges. The used sponges were collectedfrom individuals at the O-Cel-O sponge manufacturing facility inTonawanda, N.Y., who had acquired said sponges for use in their home.These used sponges had experienced no greater than 30 days use sincetheir date of purchase, and had experienced a variety of uses throughoutthe home environment with a variety of cleaning chemicals. The fungalspores were harvested from the isolated molds which had been grown up inpure culture on PDA, and included Aspergillus niger, three undefinedAspergillus species, a Stachybotrys species, two undefined Penicilliumspecies, and an Alternaria species. The ASTM designated species includedAspergillus niger, Penicillium funiculosum, Chaetomium globosom,Aerobasidium pullulans, and Cliocladium virens. The sponge samples werecut into 2.54 cm square pieces, and were placed in duplicate intominimal salts agar (M9) in a petri dish. The M9 agar contained essentialmineral ions necessary for growth, but no carbon source. The sponges,when placed into the agar, became the sole carbon source for any moldgrowth seen on the plates after inoculation. The mold suspension wasprepared by placing 50 μl of a 10⁸ spore/ml suspension into sterile M9broth. For this ASTM method, equal numbers of spores were blended toyield a final total spore concentration of 5·10⁵ spores/ml for both theASTM designated organisms and the isolates. Spray inoculation wascarried out in accordance with the procedure for the Potato DextroseAgar activity test described in Comparative Example 29 until the surfaceof the sponges was damp. The plates were closed and incubated at 28C for28 days at 95% relative humidity. The plates were checked daily for odorproduction and by microscopy for visual growth. Results from these testsare presented in Table XVII, where data show the days to failure afterinoculation due to mold growth, days to failure after inoculation forodor production, and the final ASTM rating of the samples after the 28day test duration. From the data it is generally observed that odorproduction preceded visual detection of growth (failure) byapproximately one day, and thus visual detection might be considered agood measure of odor production. The rating system was as follows:

    ______________________________________                                        ASTM Rating Meaning                                                           ______________________________________                                        0           No growth of mold detected                                        1           1-10% sample coverage by mold growth                              2           10-30% sample coverage                                            3           30-60% sample coverage                                            4           >60% sample coverage                                                          (total sample failure)                                            ______________________________________                                    

The data in Table XVII indicate that the ASTM and isolate moldsflourished on the quaternary ammonium treated sponges, even for thecases without rinsing. The ASTM molds had only limited growth on thechitosan-zinc-pyrithione sponges, while the isolate molds showed zerogrowth on the same for all rinse cases. The data for the spongecontaining ZnO indicates activity against molds is similar to the casewithout ZnO, although there appears to be some elevation in activity forthe ZnO containing sponge at the intermediate 50x sponge rinsingtreatment. The results confirm the superior activity and durability ofthe chitosan-zinc-pyrithione treatment compared to commonly employedquaternary ammonium treatment of commercially available sponge.

                  TABLE XVI                                                       ______________________________________                                                                   Log     Log                                                         Water     Reduction                                                                             Reduction                                  Ex.    Treatment*                                                                              Rinses    Immediate                                                                             Final                                      ______________________________________                                        30A    Quaternary                                                                              none      -0.16   6.43                                       30B    Quaternary                                                                              20        -0.39   -0.46                                      30c    Quaternary                                                                              50        -0.42   2.36                                       30D    Quaternary                                                                              100       -0.37   2.43                                       31A    ZP        none      0.39    6.35                                       31B    ZP        20        0.33    4.91                                       31C    ZP        50        0.36    -0.39                                      31D    ZP        100       0.36    -0.42                                      32A    ZP + ZnO  none      0.49    6.35                                       32B    ZP + ZnO  20        0.36    6.35                                       32C    ZP + ZnO  50        0.49    6.35                                       32D    ZP + ZnO  100       0.49    3.19                                       ______________________________________                                         *Quaternary implies treated with quaternary ammonium solution described i     Precursor 3; ZP implies containing chitosanzinc-pyrithione complex.      

                  TABLE XVII                                                      ______________________________________                                        RESULTS FROM MOLD RESISTANCE                                                  TESTING FOR SPONGES                                                           ASTM Designated     Isolated Sponge                                           Organisms           Organisms                                                                Final                Final                                     Days to Failure                                                                              ASTM     Days to Failure                                                                           ASTM                                      Sample                                                                              Odor    Growth   Rating Odor Growth Rating                              ______________________________________                                        30A   3       6        4      23   24     4                                   30B   3       6        4      3    6      4                                   30C   3       6        4      3    7      4                                   30D   3       6        4      6    7      4                                   31A   >28     >28      0      >28  >28    0                                   31B   25      27       3      >28  >28    0                                   31C   14      15       4      >28  >28    0                                   31D   14      15       4      >28  >28    0                                   32A   >28     >28      0      >28  >28    0                                   32B   26      27       4      >28  >28    0                                   32C   19      18       3.5    >28  >28    0                                   32D   13      15       4      >28  >28    0                                   ______________________________________                                    

Example 33

A sponge block was prepared in accordance with the procedure describedin Example 31. Sponge samples were cut and further treated as alsodescribed in Example 31 except instead of employing sodium pyrithione,potentiation of the sponge was carried out using NACAP. In this case,potentiation of four sponges in a plastic bag was carried out byaddition of a solution comprised of 21.3 g of sodiummercaptobenzothiazole solution (50%) diluted with 949 g DI water. Thesesponges exhibited a strong yellow coloration upon contact with the NaMBTsolution indicating chelation of mercaptobenzothiazole with thechitosan-zinc complex in the sponge. Sponges were then subjected to therinsing treatments described in Comparative Example 30. The yellowcoloration persisted even up to 100 rinses which indicated that thechitosan-zinc-mercaptobenzothiazole complex in the sponge was durable tosuch rinsing.

Example 34

Sponge samples were taken from a sponge block prepared as described inExample 31 and were further treated as also described in Example 33except instead of employing sodium pyrithione, potentiation of thesponge was carried out using sodium dimethyldithiocarbamate (sold asAquatreat SDM, available from the Alco Chemical Company, Chattanooga,Tenn.). In this case, potentiation of four sponges in a plastic bag wascarried out by addition of a solution comprised of 20.2 g of sodiumdimethyldithiocarbamate solution (40%) diluted with 948 g DI water.These sponges exhibited no discoloration of the sponge upon contact withthe potentiation solution. Sponges were then subjected to the rinsingtreatments described in Comparative Example 30. No change in appearancewas observed even after 100 rinses.

In summary, a novel and unobvious anti-microbial composition and articleincorporating the anti-microbial agent have been described. Althoughspecific embodiments and examples have been disclosed herein, it shouldbe borne in mind that these have been by way of explanation andillustration and the present invention is not limited thereby. Certainlymodifications which are not within the ordinary skill in the art areconsidered to lie within the scope of this invention as defined by thefollowing claims.

I claim:
 1. A substantially water insoluble metal complex comprising:a.at least one chelating polymer selected from the group consisting ofpolyglucosamine, polycarboxylic acid, polyamine and ethylene acrylicacid copolymer; b. one or more metal ions chelated to said chelatingpolymer said metal ions selected from the group consisting of atransition metal ions, non-transition metal ions capable of existing inmore than one valence state, zinc and aluminum; and c. at least onepotentiator chelated to said transition metal ion.
 2. The complex ofclaim 1 wherein said transition metal ion is selected from the groupconsisting of zinc, zirconium, copper and aluminum.
 3. The complex ofclaim 1 wherein said chelating polymer is chitosan.
 4. The complex ofclaim 1 wherein said potentiator is selected from the group consistingof an imidazole, a pyrithione, a thiazole and an alkyl dithiocarbamate.5. The complex of claim 1 further including zinc oxide.
 6. Asubstantially water insoluble metal complex comprising:a. at least onechelating polymer consisting of chitosan; b. one or more metal ionschelated to said chelating polymer said metal ions selected from thegroup consisting of a transition metal ions, non-transition metal ionscapable of existing in more than one valence state, zinc and aluminum;and c. at least one potentiator chelated to said transition metal ion.7. The complex of claim 6 wherein said potentiator is selected from thegroup consisting of an imidazole, a pyrithione, a thiazole and an alkyldithiocarbamate.
 8. The complex of claim 6 further including zinc oxide.